Tumor mutation burden involving epigenetic regulatory genes and the RhoA GTPase predicts overall survival in nodal mature T-cell lymphomas

Nodal mature T-cell lymphomas (nMTCL) comprises a heterogeneous group of rare malignancies with aggressive biological behavior and poor prognosis. Epigenetic phenomena, including mutations in genes that control DNA methylation and histone deacetylation, in addition to inactivating mutations in the RhoA GTPase, play a central role in its pathogenesis and constitute potential new targets for therapeutic intervention. Tumor mutational burden (TMB) reflects the process of clonal evolution, predicts response to anti-cancer therapies and has emerged as a prognostic biomarker in several solid neoplasms; however, its potential prognostic impact remains unknown in nMTCL. In this study, we conducted Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) diagnostic tumor samples using a target-panel to search for recurrent mutations involving the IDH-1/IDH-2, TET-2, DNMT3A and RhoA genes in 59 cases of nMTCL. For the first time, we demonstrated that high-TMB, defined by the presence of ≥ two mutations involving the aforementioned genes, was associated with decreased overall survival in nMTCL patients treated with CHOP-like regimens. Additionally, high-TMB was correlated with bulky disease, lower overall response rate, and higher mortality. Future studies using larger cohorts may validate our preliminary results that indicate TMB as a potential molecular biomarker associated with adverse prognosis in nMTCL.


Introduction
Mature T-cell lymphomas (MTCL) represents a heterogeneous group of post-thymic T-cell malignant neoplasms that are highly aggressive and frequently resistant to chemotherapy based on anthracyclic agents. Patients with MTCL have an estimated 5-year overall survival (OS) around 30-40% [1,2]. It accounts for 15% of all non-Hodgkin's lymphomas (NHL), and according to the 2016-World Health Organization (WHO) Classification of Hematopoietic and Lymphoid Tissue Neoplasms, are categorized as predominantly nodal, extra nodal, primary cutaneous and disseminated or leukemic [1]. Although Page 2 of 8 de Pádua Covas Lage et al. Clinical Epigenetics (2022) 14:180 rare, these tumors show characteristic geographic distribution, being more prevalent in East Asia and South America, where it represents up to 25% of all NHL [1,2]. The nodal subgroup (nMTCL) encompasses approximately 60-70% of MTCL cases, comprising four main histopathological variants, including peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS); angioimmunoblastic T-cell lymphoma (AITL) and other nodal MTCL with T-helper follicular phenotype (nMTCL-THf ), such as the follicular T-cell lymphoma with t(5;9) (q33;q22)-ITK/SYK rearrangement; and ALK1 (anaplastic lymphoma kinase-1) positive and negative systemic anaplastic large cell lymphomas (sALCL) [1]. Commonly, patients with nMTCL present unfavorable biological features at diagnosis, including inactivation of the tumor suppressor genes TP53 and p15INK4b/ p16INK4a, high concentration of P-glycoprotein (Pgp) into neoplastic cells and multi-drug resistance phenotype (MDR), which explain the high rates of therapeutic failure with CHOP-like (cyclophosphamide, doxorubicin, vincristine, and prednisone) regimens [2].
The RhoA gene (Ras homolog family member A), located on chromosome 3, encodes a small GTPase that controls the conformation of the cytoskeleton, T-cell receptor (TCR) signaling, and plays a central role in the ontogenesis of T-lymphocytes [7]. Yoo et al. [8] identified recurrent mutations involving the RhoA gene in nMTCL-THf patients, particularly in AITL. The RhoA G17V point mutation, associated with loss of function of this GTPase, is closely linked to nMTCL-THf oncogenesis, occurring in up to 60% of AITL cases [7,8]. Currently, RhoA mutations are considered as diagnostic biomarkers of AITL and its correlated disorders; however, their prognostic role remains controversial [8][9][10].
Tumor mutation burden (TMB) has emerged as a prognostic biomarker in several solid tumors such as breast, prostate, and lung cancers [11]. High TMB has been associated with better responses to anti-cancer therapies and correlates with clinical outcomes in different malignancies. In this sense, TMB also has been considered a promising response biomarker to immune checkpoint inhibitors [12,13]. Furthermore, TMB reflects the clonal evolution, characterized by progressive addition of molecular-genetic abnormalities, as well as intratumor heterogeneity [14]. Recently, Falchi et al. reported a correlation between high TMB on epigenetic regulatory genes and higher overall response rate (ORR) to 5-azacytidine and romidepsin in nodal MTCL [5]. However, the potential prognostic of epigenetic TMB remains largely unknown in nodal MTCL. Based on this premise, herein we aimed to establish a correlation between epigenetic TMB and prognosis in nodal MTCL, as well as searching for associations between TMB and clinical phenotype in this subgroup of neoplasms.

Methods
We conducted a retrospective, observational and singlecenter study involving 59 patients with biopsy-proven diagnosis of nodal MTCL, treated at the Department of Hematology, University of São Paulo, Brazil, from January 2000 to December 2019. The study was approved by the Research Ethics Committee of our institution (number 02975012.0.0000068) and was allowed to waive the application of the Informed Consent Form. Clinical, laboratory and epidemiological data were captured from electronic medical records and Database of the NHL Group at the University of São Paulo.
All patients were tested for HIV and HTLV-1 during staging procedures, and those with positive serology were excluded. Likewise, non-systemic ALK-1 negative ALCL patients, such as those with cutaneous primary ALCL, lymphomatoid papulosis and ALCL associated with breast implants were also excluded, as well as those cases aged ≤ 18 years old. All cases underwent blood counts, biochemical tests, including kidney and liver function, serum lactate dehydrogenase (LDH) dosage, transthoracic echocardiography to estimate myocardial function pre-exposure to anthracyclines, imaging tests, including computed tomography (CT) of the neck, chest, abdomen and pelvis and/or 18-FDG-PET CT, as well as unilateral bone marrow biopsy. Selected cases underwent neuroimaging examinations, including brain magnetic resonance imaging (MRI), cerebrospinal fluid analysis, as well as endoscopic evaluation of the gastrointestinal tract.
For cases suggestive of AITL/nMTCL-THf, IHC study incorporating the monoclonal antibodies for For statistical analysis, only non-synonymous mutations were considered. Survival curves were constructed using the Kaplan-Meier method and the Log-Rank test was used to establish correlation between TMB, overall survival (OS) and progression-free survival (PFS). Chisquare test was used to establish a relationship between TMB or recurrent mutations and clinical-laboratorial characteristics. Statistical tests were performed using the software STATA 12.0 and a p value < 0.10 was considered statistically significant.
As summarized in Table 1

Discussion
In this study, we demonstrated for the first time that high-TMB, involving epigenetic regulatory genes and the RhoA GTPase, adversely affected the OS of patients with nodal MTCL. In our cohort, patients with ≥ 2 mutations involving the IDH-1/IDH-2, DNMT3A, TET-2 and/ or RhoA genes, accessed by Sanger sequencing in diagnostic FFPE biopsies, had an estimated 2-year OS of only 36.4% versus 64.3% for cases with less than 2 mutations, p = 0.08. Similarly, we were able to establish an association between a high number of mutations involving these epigenetic modifiers and unfavorable clinical phenotype in nodal MTCL. Patients with high-TMB showed statistically significantly higher tumor volume, represented by bulky disease ≥ 7 cm (p = 0.01), as well as higher mortality (p = 0.04) and elevated failure rate to anthracyclinebased chemotherapy (p = 0.08) compared to those cases with low-TMB.
Due to the frequent overlapping of clinical, pathological, phenotypic and molecular-genetic findings among the four main histological subtypes of nodal MTCL recognized by the latest WHO Classification of Hematopoietic Neoplasms (WHO-2016) and taking into account the high rate of therapeutic failure with conventional chemotherapy regimens, the discovery of new diagnostic and prognostic biomarkers is fundamental [2,15]. In this sense, our group recently demonstrated that peripheral monocyte count ≥ 1.5 × 10 9 /L at diagnosis, overexpression of the CCNA2 gene and CHEK1 protein, high tissue expression of the GATA-3 gene and mutations involving the TET-2 gene may be potential biomarkers capable of assisting in the diagnostic discrimination of the different nodal MTCL histological subtypes. These biomarkers were also capable of predicting adverse clinical outcomes for MTCL [16][17][18][19].
Such new genetic-molecular biomarkers may also represent specific targets for therapeutic intervention. The high concentration of P-glycoprotein (Pgp) in MTCL tumor cells, as well as the frequent inactivation of tumor suppressor genes, such as TP53 and p15INK4b/p16INK4a, confer a multidrug resistance phenotype (MDR), thus explaining the poor results achieved with regimens based on anthracyclic agents and vinca alkaloids in the up-front therapy of nodal MTCL [2,[20][21][22][23]. Aiming to improve the therapeutic results, new target-drugs have been considered for the management of nodal MTCL, specifically for cases with follicular T-helper (THf ) origin. In this sense, the incorporation of the anti-CD30 immunoconjugate brentuximab vedotin and the use of epigenetic modifiers based regimens, such as the association CHOP plus 5-azacytidine or the combination of 5-azacytidine plus romidepsin, have been shown to be safe and effective in the management of ALCL and nMTCL-THf, respectively [5,6,24,25]. Several studies firstly conducted in myeloid neoplasms, such as myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), appointed to the recurrence of TET-2 mutations in these tumors and correlate such mutations with an adverse phenotype, as well as with responsiveness to HMAs [26][27][28][29]. TET-2 mutations, together with other mutations involving epigenetic regulatory genes also play a central role in the oncogenesis of nodal MTCL-THf, and different groups associate their occurrence with greater biological aggressiveness and poor outcomes in these tumors [19,30].
Based on this biological knowledge, Falchi et al. [5] conducted a phase 2 trial involving 25 patients with nodal MTCL up-front treated with the epigenetic modifiers 5-azacytidine and romidepsin. In that study, tumor samples were submitted to next-generation sequencing (NGS) to search for mutations involving epigenetic regulators. The authors demonstrated that responders, particularly those with the THf-phenotype, had a greater mutational burden in genes involved in DNA methylation and histone deacetylation, suggesting that high TMB involving epigenetic modifiers may be a predictor of therapeutic response to HMAs and HDACi in nodal MTCL [5]. However, due to the small sample size (N = 25), these authors were unable to assess the potential prognostic impact of epigenomic TMB on nodal MTCL, different to what was demonstrated by our group. On the other hand, as our cohort was primarily treated with CHOP-like regimens, we could not test association between the number of mutations involving epigenetic regulators and responsiveness to HMAs and HDACi.
Similarly to Falchi et al. [5], other research groups were also able to establish an association between high TMB and therapeutic susceptibility to different anti-cancer agents in solid cancers. Studies involving patients with prostate, breast, lung, pancreas tumors and high-grade gliomas demonstrate the ability of high TMB to predict response to immunotherapy, including PD-1/PD-L1 inhibitors [31][32][33]. Recently, the immunecheckpoint inhibitor pembrolizumab was approved by the FDA for the treatment of adults and children with advanced cancers, who have a high TMB, defined as ≥ 10 mutations/megabase of tumor DNA after failure of other chemotherapeutic agents.
A limitation related to our study refers to the restricted gene panel tested. Next-generation sequencing with broader gene panels, particularly whole-exome sequencing (WES), might be able to more reliably estimate the TMB, through genome-wide scanning and the construction of an index contemplating the number of mutations found per DNA mega-bases (mut/ Mb). Although we used the Sanger technique, our panel incorporated the main regulators involved in the biology of nodal MTCL and included primers designed to access the main recurrent mutations described in these malignancies [8,[34][35][36].

Conclusion
In conclusion, to our knowledge, these results showed for the first time that high TMB, defined here by the presence of two or more mutations involving epigenetic regulatory genes and the RhoA GTPase, was associated with decreased OS in patients with nodal MTCL. High epigenetic TMB was also correlated with adverse parameters, such as bulky disease, decreased ORR for CHOPlike regimens and higher mortality, and may represent a predictor of response to HMA and HDACi, as previously suggested by other groups. Although preliminary and lacking external validation, these data highlight the potential of epigenetic/GTPase TMB as an important prognostic biomarker in nodal MTCL.